Part:BBa_K4632003:Experience

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Applications of BBa_K4632003

1. Verifying the Expression and Secretion Proficiency of CPTI

The OmpA -CPTI fragment (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) was inserted into the pET-30a vector to generate the plasmid pET-30a-OmpA-CPTI. This plasmid was then transformed into E. coli BL21 and cultured overnight in LB medium. Overnight culture was dilute by 1/1000 to fresh LB medium, the IPTG was added when the OD600 reach 0.6. The medium was incubated another 3 hours after the ITPG addition, then centrifuged at 12,000 rpm 10 mins to obtain the supernatant and the precipitate. The supernatant was concentrated using ultrafiltration centrifuge tubes, while the precipitate was subjected to cell lysis with a lysis buffer. After lysis, the sample was centrifuged again to separate the supernatant from the post-lysis precipitate. The post-lysis precipitate was resuspended in lysis buffer. Tricine-PAGE (Figure 3) and Western Blot analyses (Figure 4) were performed on the above samples, and the results indicated that the CPTI protein was not expressed in E. coli.

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Figure 3. Tricine-PAGE analysis of CPTI expression. <p>Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysateof of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

Figure 4: Western blot analysis of CPTI expression <p>Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysate of of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

With IPTG the cell could produce the detectable CPTI (Lane 4, Figure 4), however, the concentrated supernatant of CPTI could not been seen (Lane 1, Figure 4). This might because of the poor solution of CPTI with OmpA singlal peptide. To get a extracellular expressed CPTI, the GST tag, which could improve the solution of certain protein, was successfully added at the C-terminal of the CPTI gene. The protein induction expression experiment is currently underway.

This modification ensures that the protein can be expressed to a significant extent, as similar experiments have been reported (Yang et al., 2003). Our unique approach involves testing the activity of CPTI without removing the GST tag, which distinguishes our study from previous ones.

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